Proteins for therapeutic use are currently available in suitable forms in adequate quantities largely as a result of the advances in recombinant DNA technologies. The availability of recombinant proteins has endangered advances in protein formulation and chemical modification. One goal of such modification is protein protection. Chemical attachment may effectively block a proteolytic enzyme from physical contact with the protein backbone itself, and thus prevent degradation. Additional advantages include, under certain circumstances, increasing the stability and circulation time of the therapeutic protein and decreasing immunogenicity. One such method commonly used for protein modification is by covalent attachment of water soluble polymers.
Polyethylene glycol (“PEG”) is one such chemical moiety which has been used in the PEGylation of therapeutic protein products. The US FDA has approved PEG for use as a vehicle or base in foods, cosmetics and pharmaceuticals, including injectable, topical, rectal and nasal formulations. Molecules coupled to PEG become non-toxic, nonimmunogenic, soluble in water and many organic solvents, and surfaces modified by PEG attachment become hydrophillic and protein rejecting.
The FDA has approved several PEGylated polypeptides as therapeutics and more are undergoing clinical investigation. In 1990, pegademase (Adagen) received approval for the treatment of severe combined immunodeficiency (SCID). Pegaspargase (Oncaspar) approved in 1994, contains the pegylated enzyme L-asparaginase, used clinically in combination with chemotherapy for the treatment of acute lymphocytic leukaemia, acute lymphpblastic leukaemia and chronic myelogenous leukaemia. In 2001, peginterferon α2b (PegIntron) became available as a once-a-week treatment for hepatitis C. Peginterferon α2a (Pegasys) approved in 2002 used a second generation, branched PEG of 40 kDa conjugated through a ε-NH2 group of lysine used as spacer to interferon α2a increased the half life of IFN-α2a from 9 to 77 hours. A pegylated form of human growth hormone antagonist called pegvisomant (Somavert) was approved by FDA in 2003 for the treatment of acromegaly. Doxil, a pegylated liposomal formulation of doxorubicin was approved in 1995 for the treatment of Kaposi's sarcoma. Pegfilgrastim (Neulasta), approved in 2002, is a pegylated form of the earlier drug filgrastim (Neupogen) used for the treatment of neutropenia. PEGylation has taken 20 years to emerge as a viable pharmaceutical tool. Over the period there have been important advances in the chemistry of PEGylation, in the generation of biomolecule therapeutics and in understanding PEG-biomolecule conjugates. PEGylation is now established as the method of choice for improving the pharmacokinetics and pharmacodynamics of protein pharmaceuticals.
A variety of active PEGs have been prepared. mPEG succinimidyl succinate and mPEG succinimidyl carbonate were the reagents used and approved by US FDA. The reagents had the limitation of forming weak linkages between the PEG moiety and protein, potential unwanted side reactions, contamination, and restriction to low MW PEGs. The above limitations were overcome by use of mPEG-propionaldehyde which was easier to prepare. PEG aldehydes are inert toward water and react primarily with amines. Inertness toward water is desired, not only because of efficiency of storage, preparation, and application, but also because it permits stepwise linkage, in aqueous media, of molecules to surfaces and molecules to molecule. mPEG aldehyde has essentially all the properties of ideal PEG derivative i.e. reactive with nucleophillic groups (typically amino) on proteins and surfaces; stable in aqueous media and on the shelf; easily prepared and characterized; and capable of coupling to proteins without reducing protein activity.
U.S. Pat. No. 5,824,784 assigned to Amgen claims a substantially homogenous preparation of N-terminally monoPEGylated G-CSF or analog thereof and a method for attaching a polyethylene glycol to a G-CSF molecule wherein the PEG moeity has single aldehyde group. The PEGylation process claims reacting G-CSF with polyethylene glycol under reducing alkylation conditions at a pH sufficiently acidic to selectively activate the alpha amino group at the amino terminus of G-CSF. The process discloses the addition of a 5-fold molar excess of methoxypolyethylene glycol aldehyde of average MW, 6 kDa to a cooled (4° C.) stirred solution of rhG-CSF (1 ml, 5 mg/ml) in 100 mM sodium phosphate, pH 5, containing 20 mM NaCNBH3. The stirring of the reaction mixture was continued at the same temperature The mono-mPEG-GCSF derivative was purified by ion exchange chromatography using HiLoad 16/10 S SEPHAROSE HP column and eluted with a linear 400 minute gradient from 0% to 45% 20 mM sodium acetate, pH 4, containing 1M NaCl. The % composition of N terminally mono-mPEG-GCSF obtained by reductive alkylation is not disclosed. A comparative stability analysis of N-terminally momopegylated G-CSF obtained by amide linkage (derived by using N-hydroxy succinimidyl ester of carboxymethyl methoxy polyethylene glycol as nucleophile) and the other obtained by amine linkage for 8 weeks yielded 82% purity with respect to one having amine linkage between the protein and the mPEG-aldehyde. A surprising result was observed as the amine linkage produced a material with far fewer aggregates against the one with amide linkage.
The present invention discloses a simple and improved process to enhance the efficiency of pegylation process by addition of a polyol having the formula CnH2n+2On where n is from 3 to 6, or a carbohydrate, or a derivative thereof. Pursuant to following the Example 2 of U.S. Pat. No. 5,824,784, it was surprisingly found that addition of a polyol having the formula CnH2n+2On where n is from 3 to 6, or a carbohydrate, or a derivative thereof to the pegylation buffer after buffer exchange from the storage buffer to the pegylation buffer and maintenance of the concentration of said polyol having the formula CnH2+2On where n is from 3 to 6, or a carbohydrate, or a derivative thereof during the entire pegylation process not only increases the pegylation yield but also results in the formation of pure monopegylated r-metHuG-CSF with >80% purity thus minimising the formation of aggregates. Moreover the addition of polyol having the formula CnH2n+2On where n is from 3 to 6, or a carbohydrate, or a derivative thereof leads to the minimal amount of unreacted r-metHuG-CSF as compared to the process carried out in the absence of the same. Kinstler et al., has investigated the liquid stability of rhG-CSF after PEG with an average molecular weight of 6000 daltons covalently attached to the N-terminal methionine wherein the covalent attachment was effected either through alkylation and acylation. The N-terminally PEGylated rhG-CSF conjugates were purified by cation exchange chromatography. Physical characterization indicated no apparent differences in the rhG-CSF molecules that were conjugated with either method. Stability, in liquid at elevated temperatures, of these conjugated molecules indicated that the primary pathway of degradation was aggregation. Conjugation through alkylation offered the distinct advantage of decreasing, by approximately 5 times, the amount of aggregation present as compared to acylation. Therefore, it was suggested that the increased aggregation observed with the acylation conjugation method may result from the charge neutralization of the N-terminal a-amino group of rhG-CSF. The detrimental effects of aggregation in parenteral formulations of therapeutic proteins affirms the importance of minimizing this type of degradation.
Protein aggregation and subsequent deposition as insoluble fibrils or amorphous precipitates is responsible for a number of diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and systemic amyloidosis. Protein aggregation is also a dominant degradation pathway for therapeutic proteins, potentially occurring during all phases of production,purification, shipping, storage, and administration. Protein aggregates in parenterally delivered protein formulations can cause adverse reactions in patients ranging from immune responses to anaphylactic shock. There is always a need to have stable parenteral formulations by minimizing the aggregates formation affecting the purity and activity of proteins over its shelf life.
Carpenter et al., investigated the aggregation of rhGCSF, a protein that rapidly aggregates and precipitates at pH 6.9 and 37° C. It was found that native monomeric rhGCSF reversibly forms a dimer under physiological conditions and that the dimeric species does not participate in the irreversible aggregation process. Sucrose, a thermodynamic stabilizer, inhibits the aggregation of rhGCSF. Carpenter et al. had postulated that sucrose acted by reducing the concentration of structurally expanded species, consistent with the hypothesis that preferential exclusion favors most compact species in the native state ensemble.
Rajan et al., in a study conducted under physiological pH and temperature, showed that N-terminal attachment of a 20 kDa PEG moiety to GCSF had the ability to 1) prevent protein precipitation by rendering the aggregates soluble, and 2) slowed the rate of aggregation relative to GCSF.
Yun et al., disclosed a novel mPEG derivative, containing a reaction group of 1-methyl pyridinium toluene-4-sulfonate conjugated to rhGCSF and consensus interferon to obtain homogeneous mono-PEGylated proteins which were identified by high performance size-exclusion and MALDI-TOF mass spectrometry.
Aggregation is agglomeration of proteins that frequently is irreversible when introduced into physiologic fluids, leading to inactivation or increased immunogenicity. Aggregation is a common problem with protein pharmaceuticals and may compromise process isolation yields, limit shelf life, cause failure in manufacturing, and prevent applications to new advances in delivery. Exposure of proteins to shear, agitation, and multiple surfaces is unavoidable and may induce aggregation.
Protein concentration is an important variable for ameliorating aggregation. The initial conformation related reaction leading to aggregation is expected to be first order but the subsequent aggregation of nonnative states is expected to be a second or higher order process because the frequency of collisions varies with concentration. Therefore, aggregation is expected to accelerate with increased protein concentration. Sorbitol (D-glucitol) is a polyol commonly used as an excipient in liquid parenteral biologic formulations and even as a food sweetening agent. Sorbitol provides effective protein stabilization in the liquid state and several marketed biologics are formulated in sorbitol including Neulasta and Neupogen.
Carbohydrates such as sucrose, glucose, mannose, and trehalose as well as polyhydric alcohols like glycerol, sorbitol, and mannitol have frequently also been used to enhance the solubility of proteins. This effect is presumably mediated through a combination of mechanisms including preferential hydration effects and increase in solvent surface tension as well as weak interactions with the surface of the proteins. The excellent biocompatibility of these compounds make them of general utility in this regard since little effect on protein structure and activity is usually seen in the presence of high concentrations of polyols, especially carbohydrates. The protein solubility problem is in many ways analogous to the protein folding problem in that very small differences between complex thermodynamic states account for the phenomena. It follows that an accurate description of the two critical states of interest, the structure of the surface hydration shell of the protein and the nature of the intermolecular contacts in the solid phase, is necessary to quantitatively account for the solubility of a particular macromolecule. Alteration of solubility using external variables suggest that only minor alterations of the solvent or solute should be sufficient to perturb protein solubility. Addition of physiologically acceptable compounds such as salts, sugars, and amino acids can be used to control protein solubility in an empirical manner. Since these same agents will sometimes enhance protein stability, the right combination of circumstances could result in a single compound providing stabilizing, solubilizing, and buffering capacity.
In addition to the above, use of polar organic solvents for enhancing the pegylation efficiency are already known in the prior art. PCT publication no. WO 02/28437 disclosed liquid-phase pegylation of growth hormone releasing factor, which allows to obtain regioselectively GRF-PEG conjugate having 1 PEG molecule covalently bound to the ε-amino group Lys12 characterized in that the reaction was carried out in a structuring solvent specifically alcohol and more specifically trifluoroethanol. The advantages cited were higher yields and the scaleability of the pegylation process.
Another PCT publication no. WO 2008/051383 A2 disclosed a method of producing a composition of matter wherein the method involved obtaining a pharmacologically active peptide, and conjugating the peptide to a pharmacologically acceptable PEG by reacting the peptide with a PEG-aldehyde compound at a free amine moiety on the peptide in a buffer solution comprising an alcohol co-solvent. The use of the method is particularly useful for pegylating peptides that are relatively insoluble in an aqueous medium, typically peptides with aqueous solubility below about 0.1 to 10 mg/ml. Additional benefits included acceleration of pegylation reaction and improved pegylation efficiency. The pegylation efficiency by reductive amination of peptide sequences benefits from the use of more hydrophobic alcohols and the efficiency enhancement afforded by fluoro alcohols was most advantageous to reductive amination reactions. The % of alcohols found to be beneficial was in the range of 30% to 70% v/v.
WO 2008/051383 A2 exemplified usage of Isopropyl alcohol(IPA), Trifluoroethanol (TFE), and hexafluoro-isopropyl alcohol (HFIPA) as co-solvents for enhancement in pegylation efficiency showing increased product yields for pegylating Calcitonin gene related peptides (CGRP) that were relatively insoluble in an aqueous medium, typically peptides with aqueous solubility below about 0.1 to 10 mg/ml. The mono-PEGylated peptide product was quantitated by integration of RP-HPLC chromatograms and reported as % product peak. 50% IPA and 50% TFE surprisingly showed 2.6 fold increase in product yield with IPA and 4.1 fold with TFE respectively. The PEGylation reaction yields in almost 42 CGRP peptides tested showed the reaction yields between about 50% to about 70% as against of less than 20% seen in the absence of the alcohol co-solvent. A variety of alcohol co-solvents were tested in the buffer solution for the conjugation reaction in an attempt to solubilize less soluble peptide and to improve conjugate yields.
The above prior art are applicable to the synthetic peptides produced by solid phase peptide synthesis or solution phase synthesis. However the harsh conditions when alcohols are used at such high concentration cannot be used for pegylating proteins. Moreover the PEGylation was carried out at concentration of 2 mg/ml in an amine free buffer (20 mM sodium phosphate, pH 6.0) whereas in case of proteins like rhG-CSF the pegylation has to be carried out at least at a concentration of 5 mg/ml and an acidic pH sufficient to drive the reaction to completion. Hence an essential element of the present invention is use of non-hazardous additives for increasing the pegylation efficiency specially of proteins. Also an inherent limitation of the prior art is the handling of volatile and hazardous alcohols limits the use for large scale operations. An essential element of the instant invention is surprising effect of increased pegylation efficiency and product yield by addition of a polyol or a carbohydrate or a derivative thereof to pegylation buffer in presence of a suitable reducing agent by keeping the r-metHuG-CSF in solubilized form. Another element of the instant invention is to develop a cost-effective and robust process for gram scale production of PEG-r-metHuG-CSF wherein the pegylation is carried out in the storage buffer of r-metHuG-CSF by just concentrating the protein by addition of polyol having the formula CnH2n+2On where n is from 3 to 6, or a carbohydrate, or a derivative thereof, by adjusting the pH of the reaction medium to drive the pegylation reaction and conjugating the mPEG aldehyde to r-metHuG-CSF wherein the process is less time intensive and eliminates the initial step of buffer exchange. Still another element of the instant invention is reduction in use of stoichiometric molar ratio of r-metHuG-CSF to PEG from 1:5 to 1:2.5 wherein the cost reduction achieved is 1.5 fold over the process using 5 molar excess of PEG reagent. Another important element of the invention is eluting the monoPEGylated r-metHuG-CSF in the presence of polyol or carbohydrate or a derivative thereof using a salt gradient in the range of 0-500 mM and concentrating the pooled monoPEGylated r-metHuG-CSF against storage buffer consisting essentially of polyol or carbohydrate or a derivative thereof and a non ionic surfactant characterized in that the purity of concentrated monoPEGylated r-metHuG-CSF is ≧99%.